京都大学 / 新制・課程博士 / 博士(医学) / 甲第24781号 / 医博第4973号 / 新制||医||1066(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 中島 貴子, 教授 溝脇 尚志, 教授 武藤 学 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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Tropomiosina (Tm) é uma das proteínas que compõe o filamento fino (actina, Tm, Troponina) do sistema muscular esquelético e desempenha um importante papel na regulação da contração muscular. Tm é um coiled-coil de 284 resíduos que forma longos homopolímeros lineares através da sobreposição de onze resíduos entre os terminais de Tms adjacentes (Interação cabeça-cauda) em condições de baixa força iônica. A presença de vários resíduos carregados (D2, K5, K6, K7, D275, H276 e D280) nas extremidades da Tm sugere que contatos intermoleculares eletrostáticos entre estes aminoácidos podem ter um importante papel na estabilidade dos polímeros. Entretanto, a estrutura do complexo cabeça-cauda demonstra que a maioria dos contatos intermoleculares na interface é de natureza hidrofóbica. A fim de analisarmos a contribuição dos grupos carregados para a estabilidade do complexo cabeça-cauda, construímos fragmentos recombinantes correspondentes à metade amino (ASTm1-142 ) e carboxi (Tm143-284(5OHW269)) terminais da proteína contendo mutações pontuais daqueles resíduos para alanina, e adicionalmente H276 para Glu. Medimos a afinidade entre todas as possíveis combinações destes fragmentos na ausência e presença de íons Mg2+, visto que este cátion está sempre presente em condições fisiológicas e é importante para estabilizar a interação entre Tm e actina. Os efeitos das mutações foram analisados por simulações de docking, desnaturações térmicas e ciclos de duplos mutantes. Os resultados demonstram que os aminoácidos K5, K7 e D280 presentes na interface formam contatos intermoleculares essenciais para a estabilidade do complexo. Enquanto, D2, K6, D275 e H276 não participam na formação de contatos intermoleculares, no entanto, contribuem para a estabilidade da interação cabeça- cauda através de suas interações intramoleculares que atuam na estabilidade das hélices individuais. Os aumentos na estabilidade da metade C-terminal da Tm (Tm143-284(5OHW)) induzidos por Mg2+ foram dependentes das mutações neste trecho da proteína sugerindo a presença de um sítio de ligação para este íon na extremidade carboxi terminal da molécula no trecho que forma a interação cabeça- cauda. Construímos um fragmento menor do C-terminal (Tm259-284(W269)) para acompanharmos mudanças no deslocamento químico induzidas pela ligação do íon usando ressonância magnética nuclear. Os resultados obtidos comprovaram nossa hipótese e nos permitiram definir pela primeira vez que a estrutura da Tm tem um ou mais sítios de ligação Mg2+ em uma região próxima ao resíduo H276 que está localizado entre vários resíduos carregados negativamente que participam da interação cabeça-cauda. Por último, estudamos os efeitos de solventes cosmótropicos (TFE e glicerol) nas estabilidades dos fragmentos da Tm, uma vez que a instabilidade (flexibilidade) da extremidade C-terminal é importante para a formação do complexo cabeça-cauda. Observamos que TFE, porém não glicerol, reduziu a afinidade entre os terminais. Ambos os co-solventes induziram aumentos na estabilidade dos fragmentos, no entanto, apenas TFE induziu um aumento no conteúdo de α-hélice e causou uma redução significativa na cooperatividade de desenovelamento das proteínas. Estes resultados indicam que estes compostos orgânicos estabilizam as estruturas dos fragmentos individuais da Tm de maneiras diferentes e que estas diferenças podem estar relacionadas aos diferentes efeitos observados na formação da interação cabeça-cauda. / Tropomyosin (Tm) is a protein component of the skeletal muscle thin filament (actin, Tm, Troponin) which has an important role in the regulation of muscle contraction. Tm is a dimeric coiled-coil (284 aminoacids) which forms long linear homopolymers through the overlap of eleven residues of adjacent Tm termini (Head- to-tail interaction) in low ionic strength conditions. The presence of several charged amino acids (D2, K5, K6, K7, D275, H276 e D280) in Tm extremities suggests that electrostatic contacts among those residues may have an important role in the stability of the polymers. Nevertheless, the solution structure of the head-to-tail complex demonstrated that most of the contacts in the interface are hydrophobic. In order to study the contribution of these charged residues to the stability of the head- to-tail complex, we built recombinant fragments corresponding to the amino (ASTm1-142) and carboxy (Tm143-284(5OHW269)) termini containing single mutations of those amino acids to alanine, and additionally a substitution of H276 for Glu. We measured the binding affinities among all possible combinations of wild-type and mutant fragments in the absence or presence of Mg2+ ions. This cation is always physiologically present in the muscle and it is known to strengthen the binding of Tm to actin. The effects of the mutations were analyzed by protein-protein docking, thermodynamic cycles and thermal denaturations. The results show that residues K5, K7 and D280 are essential to the stability of the complex. Though D2, K6, D275 and H276 are exposed to the solvent and do not participate in intermolecular contacts in the NMR structure, they may contribute to the complex stability by modulating the stability of the helices at the Tm termini. Mg2+-induced increases in stability of the C- terminal were sensitive to mutations in residues located in the head-to-tail overlap region, suggesting that Mg2+ ions may bind specifically to the carboxy extremity of the protein. We produced a small peptide (Tm259-284(W269)) to follow amide chemical shift perturbations upon Mg2+ binding by nuclear magnetic resonance measurements. The results obtained with this peptide allowed us to define for the first time that the Tm structure has one or more Mg2+ binding sites in a region centered in the vicinity of H276 in which are located several negatively charged residues that participate in the head-to-tail interaction. We also studied the effects of kosmotropic co-solvents (TFE and glycerol) in the stability of Tm fragments, as the instability (flexibility) of the C- terminal region has been pointed as important for the formation of the head-to-tail complex. We observed that TFE, but not glycerol, reduces the affinity between the termini. Both TFE and glycerol increased the stability of the isolated N- and C- terminal fragments; however, only TFE caused an increase in the helical content and a significant reduction in the cooperativity of unfolding of the proteins. Our results show that these two co-solvents stabilize the structures of individual Tm fragments in different manners and that these differences may be related to their different effects on head-to-tail complex formation.

Tropomiosina (Tm) é uma das proteínas que compõe o filamento fino (actina, Tm, Troponina) do sistema muscular esquelético e desempenha um importante papel na regulação da contração muscular. Tm é um coiled-coil de 284 resíduos que forma longos homopolímeros lineares através da sobreposição de onze resíduos entre os terminais de Tms adjacentes (Interação cabeça-cauda) em condições de baixa força iônica. A presença de vários resíduos carregados (D2, K5, K6, K7, D275, H276 e D280) nas extremidades da Tm sugere que contatos intermoleculares eletrostáticos entre estes aminoácidos podem ter um importante papel na estabilidade dos polímeros. Entretanto, a estrutura do complexo cabeça-cauda demonstra que a maioria dos contatos intermoleculares na interface é de natureza hidrofóbica. A fim de analisarmos a contribuição dos grupos carregados para a estabilidade do complexo cabeça-cauda, construímos fragmentos recombinantes correspondentes à metade amino (ASTm1-142 ) e carboxi (Tm143-284(5OHW269)) terminais da proteína contendo mutações pontuais daqueles resíduos para alanina, e adicionalmente H276 para Glu. Medimos a afinidade entre todas as possíveis combinações destes fragmentos na ausência e presença de íons Mg2+, visto que este cátion está sempre presente em condições fisiológicas e é importante para estabilizar a interação entre Tm e actina. Os efeitos das mutações foram analisados por simulações de docking, desnaturações térmicas e ciclos de duplos mutantes. Os resultados demonstram que os aminoácidos K5, K7 e D280 presentes na interface formam contatos intermoleculares essenciais para a estabilidade do complexo. Enquanto, D2, K6, D275 e H276 não participam na formação de contatos intermoleculares, no entanto, contribuem para a estabilidade da interação cabeça- cauda através de suas interações intramoleculares que atuam na estabilidade das hélices individuais. Os aumentos na estabilidade da metade C-terminal da Tm (Tm143-284(5OHW)) induzidos por Mg2+ foram dependentes das mutações neste trecho da proteína sugerindo a presença de um sítio de ligação para este íon na extremidade carboxi terminal da molécula no trecho que forma a interação cabeça- cauda. Construímos um fragmento menor do C-terminal (Tm259-284(W269)) para acompanharmos mudanças no deslocamento químico induzidas pela ligação do íon usando ressonância magnética nuclear. Os resultados obtidos comprovaram nossa hipótese e nos permitiram definir pela primeira vez que a estrutura da Tm tem um ou mais sítios de ligação Mg2+ em uma região próxima ao resíduo H276 que está localizado entre vários resíduos carregados negativamente que participam da interação cabeça-cauda. Por último, estudamos os efeitos de solventes cosmótropicos (TFE e glicerol) nas estabilidades dos fragmentos da Tm, uma vez que a instabilidade (flexibilidade) da extremidade C-terminal é importante para a formação do complexo cabeça-cauda. Observamos que TFE, porém não glicerol, reduziu a afinidade entre os terminais. Ambos os co-solventes induziram aumentos na estabilidade dos fragmentos, no entanto, apenas TFE induziu um aumento no conteúdo de α-hélice e causou uma redução significativa na cooperatividade de desenovelamento das proteínas. Estes resultados indicam que estes compostos orgânicos estabilizam as estruturas dos fragmentos individuais da Tm de maneiras diferentes e que estas diferenças podem estar relacionadas aos diferentes efeitos observados na formação da interação cabeça-cauda. / Tropomyosin (Tm) is a protein component of the skeletal muscle thin filament (actin, Tm, Troponin) which has an important role in the regulation of muscle contraction. Tm is a dimeric coiled-coil (284 aminoacids) which forms long linear homopolymers through the overlap of eleven residues of adjacent Tm termini (Head- to-tail interaction) in low ionic strength conditions. The presence of several charged amino acids (D2, K5, K6, K7, D275, H276 e D280) in Tm extremities suggests that electrostatic contacts among those residues may have an important role in the stability of the polymers. Nevertheless, the solution structure of the head-to-tail complex demonstrated that most of the contacts in the interface are hydrophobic. In order to study the contribution of these charged residues to the stability of the head- to-tail complex, we built recombinant fragments corresponding to the amino (ASTm1-142) and carboxy (Tm143-284(5OHW269)) termini containing single mutations of those amino acids to alanine, and additionally a substitution of H276 for Glu. We measured the binding affinities among all possible combinations of wild-type and mutant fragments in the absence or presence of Mg2+ ions. This cation is always physiologically present in the muscle and it is known to strengthen the binding of Tm to actin. The effects of the mutations were analyzed by protein-protein docking, thermodynamic cycles and thermal denaturations. The results show that residues K5, K7 and D280 are essential to the stability of the complex. Though D2, K6, D275 and H276 are exposed to the solvent and do not participate in intermolecular contacts in the NMR structure, they may contribute to the complex stability by modulating the stability of the helices at the Tm termini. Mg2+-induced increases in stability of the C- terminal were sensitive to mutations in residues located in the head-to-tail overlap region, suggesting that Mg2+ ions may bind specifically to the carboxy extremity of the protein. We produced a small peptide (Tm259-284(W269)) to follow amide chemical shift perturbations upon Mg2+ binding by nuclear magnetic resonance measurements. The results obtained with this peptide allowed us to define for the first time that the Tm structure has one or more Mg2+ binding sites in a region centered in the vicinity of H276 in which are located several negatively charged residues that participate in the head-to-tail interaction. We also studied the effects of kosmotropic co-solvents (TFE and glycerol) in the stability of Tm fragments, as the instability (flexibility) of the C- terminal region has been pointed as important for the formation of the head-to-tail complex. We observed that TFE, but not glycerol, reduces the affinity between the termini. Both TFE and glycerol increased the stability of the isolated N- and C- terminal fragments; however, only TFE caused an increase in the helical content and a significant reduction in the cooperativity of unfolding of the proteins. Our results show that these two co-solvents stabilize the structures of individual Tm fragments in different manners and that these differences may be related to their different effects on head-to-tail complex formation.

<p>Immunoglobulin E (IgE)-mediated allergy occurs when our immune system causes a reaction to otherwise harmless substances (allergens). Allergens are predominantly proteins present in biological materials such as pollens, mites, animal epithelia, moulds and foods. </p><p><i>In vitro</i> tests for specific IgE antibodies usually employ an allergen source extract as an antibody capturing reagent. The proportion of allergenic molecules in these biochemically complex extracts may vary.</p><p>Recombinant allergens may be obtained in large quantities with biotechnological techniques. These proteins can be characterized biochemically and immunologically, resulting in tests with minimal batch-to-batch variation. This thesis describes different uses of recombinant allergens in component-resolved diagnostics (CRD).</p><p>In CRD, single allergenic proteins are used to establish a sensitization profile of the patient. Two timothy grass (<i>Phleum pratense</i>) pollen allergens, Phl p 11 and Phl p 4, were cloned and expressed as recombinant proteins. They were subsequently characterized and can, for example, be used in a panel for grass pollen CRD.</p><p>Single allergens may be useful as diagnostic markers for allergic sensitization. This phenomenon was studied using tropomyosin, a major allergen from the shrimp <i>Penaeus aztecus</i> (Pen a 1). The characteristics of the recombinant and natural proteins were compared. The recombinant tropomyosin was then extensively tested using specific competition for IgE binding against extracts of other crustacean species, house dust mite and cockroach.</p><p>In cases when an important allergen is missing or underrepresented in a natural extract, the corresponding recombinant allergen may be added to the extract as a spiking reagent. Previous studies have shown that latex extracts for diagnostic testing may lack the allergen Hev b 5. Recombinant Hev b 5 was expressed from a synthetic gene construct, incorporating several adaptations to enable efficient large scale production of the recombinant protein, to be used as a spiking reagent.</p>

Immunoglobulin E (IgE)-mediated allergy occurs when our immune system causes a reaction to otherwise harmless substances (allergens). Allergens are predominantly proteins present in biological materials such as pollens, mites, animal epithelia, moulds and foods. In vitro tests for specific IgE antibodies usually employ an allergen source extract as an antibody capturing reagent. The proportion of allergenic molecules in these biochemically complex extracts may vary. Recombinant allergens may be obtained in large quantities with biotechnological techniques. These proteins can be characterized biochemically and immunologically, resulting in tests with minimal batch-to-batch variation. This thesis describes different uses of recombinant allergens in component-resolved diagnostics (CRD). In CRD, single allergenic proteins are used to establish a sensitization profile of the patient. Two timothy grass (Phleum pratense) pollen allergens, Phl p 11 and Phl p 4, were cloned and expressed as recombinant proteins. They were subsequently characterized and can, for example, be used in a panel for grass pollen CRD. Single allergens may be useful as diagnostic markers for allergic sensitization. This phenomenon was studied using tropomyosin, a major allergen from the shrimp Penaeus aztecus (Pen a 1). The characteristics of the recombinant and natural proteins were compared. The recombinant tropomyosin was then extensively tested using specific competition for IgE binding against extracts of other crustacean species, house dust mite and cockroach. In cases when an important allergen is missing or underrepresented in a natural extract, the corresponding recombinant allergen may be added to the extract as a spiking reagent. Previous studies have shown that latex extracts for diagnostic testing may lack the allergen Hev b 5. Recombinant Hev b 5 was expressed from a synthetic gene construct, incorporating several adaptations to enable efficient large scale production of the recombinant protein, to be used as a spiking reagent.

A prevalência de doenças alérgicas incluindo asma e dermatite atópica aumentou nos últimos anos. A asma é uma doença crônica caracterizada por hiper-responsividade das vias aéreas e limitação variável ao fluxo aéreo, reversível espontaneamente ou com tratamento. A elevada prevalência, mortalidade e custos associados tornam a doença um importante problema de saúde pública que requer atenção. A dermatite atópica (DA) é uma doença inflamatória crônica da pele, caracterizada por prurido intenso, eritema, escoriações, liquenificação na pele entre outras características. A DA causa profundo impacto na vida do indivíduo e da família e geralmente mostra os primeiros sintomas durante a infância. Ambas as doenças podem ser associadas a sensibilização a alérgenos. Nosso grupo mostrou que entre pacientes com rinite e/ou asma alérgicos a barata a tropomiosina da barata Periplaneta americana é um alérgeno principal. Em invertebrados, a tropomiosina induz resposta IgE e reatividade cruzada entre invertebrados incluindo ácaros, barata, camarão e parasitas. No presente estudo, produzimos tropomiosina recombinante de barata (alérgeno Per a 7) com elevado grau de pureza e com quantidades mínimas de endotoxina. Em modelo experimental de asma em camundongos, com sensibilização e desencadeamento com Per a 7 recombinante, houve aumento do número de células totais, e de eosinófilos, neutrófilos e linfócitos no lavado broncoalveolar. Alérgeno Per a 7 recombinante foi também utilizado em ELISA quimérico para investigar a resposta IgE a este alérgeno em 112 pacientes com dermatite atópica. A idade dos pacientes variou de 3 a 67 anos com média de 24,9 (± 15,4) anos, com 75 indivíduos do sexo feminino (67%). Nos 112 pacientes, o SCORAD apresentou média de 43,1 (± 18,1) e foram relatadas asma e rinite em 42 e 85 indivíduos, respectivamente. Níveis de IgE total apresentaram ampla variação, de 14,2 a 63.000 UI/mL, com média geométrica de 2.193 UI/mL. A idade de início dos sintomas e o tempo de doença apresentaram médias, respectivamente, de 9,5 (± 12,9) e 15,4 (± 12,3) anos. Trinta pacientes (26,8%) apresentaram níveis detectáveis de IgE para Per a 7 recombinante, com variação de 2,3 a 3.191 UI/mL. A razão de IgE específica para Per a7/IgE total nestes pacientes variou de 0,03% a 33,8%. Dividindo os pacientes em sensibilizados e não sensibilizados ao alérgeno de barata, observamos que não houve diferença significante entre os grupos com relação à idade de início dos sintomas, tempo de doença, SCORAD, presença de rinite ou asma e níveis de IgE total. Nossos resultados mostraram que o alérgeno Per a 7 recombinante induziu resposta inflamatória com caraterísticas semelhante à observada em humanos, em modelo experimental de asma em camundongos. Frequência menor de reatividade IgE ao alérgeno Per a 7 foi observada entre pacientes com dermatite atópica em nosso meio, quando comparada à observada previamente em pacientes com asma e/ou rinite. Não houve associação entre a presença de sensibilização IgE ao alérgeno Per a 7 e a gravidade da dermatite atópica, presença de asma ou rinite, idade de início e tempo de doença, e níveis de IgE total. Entretanto, a investigação do perfil de sensibilização IgE tem importância ao se considerar o uso de imunoterapia alérgenoespecífica em pacientes com dermatite atópica. / The prevalence of allergic diseases has increased in recent years, including asthma and atopic dermatitis. Asthma is a chronic disease resulting from airway hyperresponsiveness and variable airflow limitation, reversible spontaneously or with treatment. The high prevalence, mortality, and associated costs make the disease an important public health problem which requires attention. Atopic dermatitis (AD) is a chronic inflammatory skin disease, as asthma has high prevalence, and it is characterized by intense itching, erythema, excoriations, lichenification in the skin among other characteristics. The AD causes profound impact on individual and family´s life and usually shows the first symptoms during childhood. Both diseases could be related and associated with sensitization to allergens. In the case of asthma, allergy to cockroach is well known to be related with the disease. In 1999, a Brazilian study in our group has shown that among patients with allergic rhinitis and /or asthma the tropomyosin of the American cockroach Periplaneta americana (Per a 7) is a major allergen. This protein is present in vertebrates and invertebrates, and was related to induction of IgE response and cross-reactivity against among invertebrates, including mites, cockroaches, shrimp and parasites. In the present study, we produced recombinant tropomyosin from cockroach (Per a 7 allergen) with high purity and minimal amounts of endotoxin. In an experimental model of asthma in mice, with sensitization and triggering with recombinant Per a 7, there was an increase in the number of total cells, and eosinophils, neutrophils and lymphocytes in the bronchoalveolar lavage. Per a 7 allergen was also used in chimeric ELISA to investigate IgE response to this allergen in 112 patients with atopic dermatitis. Patients\' ages ranged from 3 to 67 years, mean of 24.9 (± 15.4) years, 75 female subjects (67%). In the 112 patients, a mean of SCORAD presented 43.1 (± 18.1) and asthma and rhinitis were reported in 42 and 85 individuals, respectively. Total IgE levels varied from 14.2 to 63.000 IU / mL, with a geometric mean of 2,193 IU / mL. The age at onset of symptoms and disease time presented averages, respectively, of 9.5 (± 12.9) and 15.4 (± 12.3) years. Thirty patients (26.8%) had detectable IgE recombinant Per a 7 levels, ranging from 2.3 to 3191 IU / mL. The ratio of IgA specific for Per a7 / total IgE in these patients ranged from 0.03% to 33.8%. Dividing the patients into sensitized and not sensitized to the cockroach allergen, we observed that there was no significant difference between the groups regarding the age of onset of symptoms, disease time, SCORAD, presence of rhinitis or asthma, and total IgE levels. Our results showed that recombinant Per a 7 allergen induced an inflammatory response with characteristics similar to that observed in humans in an experimental model of asthma in mice. Minor frequency reactivity of IgE Per a 7 allergen was observed among patients with atopic dermatitis in our group, when compared to that previously observed in patients with asthma and / or rhinitis. There was no association between the presence of IgE sensitization to the Per 7 allergen and the severity of atopic dermatitis, presence of asthma or rhinitis, age at onset and disease time, and total IgE levels. However, the investigation of the IgE sensitization profile is important when considering the use of allergen-specific immunotherapy in patients with atopic dermatitis

INTRODUÇÃO: A alergia a camarão é causa comum e potencialmente grave de alergia alimentar IgE mediada, incluindo anafilaxia. Ao contrário de outras alergias alimentares, a alergia a camarão afeta predominantemente adultos e geralmente é vitalícia. Até o momento, não existe terapia específica para a alergia a camarão. Os pacientes necessitam excluir o crustáceo de sua dieta e portar adrenalina auto-injetável, para uso em reações por exposição acidental. A complexidade do perfil alergênico do camarão tem sido cada vez mais reconhecida nos últimos dez anos. A proteína muscular tropomiosina foi o primeiro alérgeno do camarão identificado e é considerada o seu principal alérgeno. Alguns estudos indicam que a tropomiosina apresenta alta especificidade na alergia a camarão, sendo apontada como uma possível e importante ferramenta diagnóstica. OBJETIVO: O objetivo deste estudo foi avaliar sensibilidade, especificidade, valor preditivo positivo e valor preditivo negativo da tropomiosina em pacientes brasileiros em restrição alimentar por suspeita de alergia a camarão. MÉTODOS: Selecionou-se 32 indivíduos com suspeita de alergia a camarão, os quais foram submetidos a testes cutâneos de leitura imediata para camarão (extrato) e \"in natura\" (cru e cozido), ácaros e barata. Também foram realizadas dosagens séricas de IgE específica para camarão, tropomiosina de camarão, ácaros e barata americana. Avaliou-se reatividade clínica a camarão através de testes de provocação oral. O camarão utilizado nos testes cutâneos e nos testes de provocação oral foi o Xiphopenaeus kroyeri (sete barbas). Foi realizado Western Blot 1 D dos pacientes alérgicos. Foram realizados cálculos de valor de corte para teste cutâneo de leitura imediata e IgE sérica específica através da utilização da curva ROC. RESULTADOS: Alergia a camarão foi confirmada em 17 pacientes. A IgE sérica específica para tropomiosina de camarão neste estudo apresentou sensibilidade de 58,8%, especificidade de 60%, valor preditivo positivo de 62,5% e valor preditivo negativo 56%. Em comparação com a IgE especifica para camarão e os testes cutâneos para camarão com extrato e \"in natura\", a IgE específica para tropomiosina apresentou a menor sensibilidade e menor valor preditivo positivo. Em sete pacientes alérgicos, a tropomiosina não foi detectada, apontando a importância de outros alérgenos do camarão, avaliados no Western blot. Os testes cutâneos apresentaram diferença estatística significativa entre alérgicos e não alérgicos e foi possível definir um ponto de corte, útil na distinção entre ambos. Os resultados dos valores de corte do teste cutâneo (média) para extrato comercial foram 5,75 mm, para camarão cru 6,75 mm e para camarão cozido 5,00 mm. Todos os pacientes são atópicos. CONCLUSÃO: A IgE específica para tropomiosina neste estudo não apresentou superioridade diagnóstica quando comparada aos testes cutâneos com camarão \"in natura\" e extrato, e a IgE específica para o camarão. Outros alérgenos, além da tropomiosina, devem ser considerados na avaliação diagnóstica para a alergia a camarão / INTRODUCTION: Shrimp allergy is a common and potentially serious cause of food allergy-mediated IgE, including anaphylaxis. Unlike other food allergies, shrimp allergy affects predominantly adults and is usually lifelong. To date, there is no specific therapy for shrimp allergy. Patients need to exclude the crustacean from their diet and carry selfinjectable adrenaline to be used if the reaction begins after accidental exposure. The complexity of the allergenic profile of shrimp has been increasingly recognized in the last ten years. The tropomyosin muscle protein was the first identified shrimp allergen and is considered its main allergen. Some studies indicate that tropomyosin presents high specificity in shrimp allergy, being pointed out as a possible and important diagnostic tool. OBJECTIVE: The objective of this study was to evaluate sensitivity, specificity, positive predictive value and negative predictive value of tropomyosin in Brazilian patients under food restriction due to the suspicion of allergy to shrimp. METHODS: Thirty-two individuals with suspected allergy to shrimp were selected, who underwent immediate skin tests for shrimp (extract) and \"in natura\" (raw and cooked), mites and cockroaches. Serum dosages of IgE specific for shrimp, shrimp tropomyosin, mites and American cockroach were also performed. Clinical reactivity to shrimp was assessed by oral challenge tests. Shrimp used in skin tests and oral challenge tests was Xiphopenaeus kroyeri (seabob). Western Blot 1 D of the allergic patients was performed. Cut-off calculations were performed for immediate-reading skin test and specific serum IgE using the ROC curve. RESULTS: Shrimp allergy was confirmed in 17 patients. Serum IgE specific for shrimp tropomyosin in this study showed sensitivity of 58.8%, specificity of 60%, positive predictive value of 62.5% and negative predictive value of 56%. Compared to shrimp-specific IgE and cutaneous shrimp skin tests with extract and \"in natura\", tropomyosin was not detected, indicating the importance of other shrimp allergens evaluated in the Western blot. The skin tests presented a statistically significant difference between allergic and non-allergic and it was possible to define a cutoff point, useful in distinguishing between both. The results of the cutoff values of the skin test (average) for commercial extract were 5.75 mm, for raw shrimp 6.75 mm and for cooked shrimp 5.00 mm. All patients are atopic. CONCLUSION: The tropomyosin-specific IgE in this study did not present diagnostic superiority when compared to cutaneous tests with in natura shrimp and extract, and shrimp specific IgE. Other allergens, in addition to tropomyosin, should be considered in the diagnostic evaluation for shrimp allergy

This research delves into idiopathic autism spectrum disorder (ASD), investigating the role of TrkB signaling pathways and BDNF regulation in the cortex. Additionally, it explores offering insights into maternal influences on mouse models. / Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by challenges in social interactions and repetitive behaviors. Prevalence of ASD is estimated to be 1 in 54 globally and is rising recently in many countries including Canada. ASD affects individuals differently, making diagnosis challenging. At present, no molecular diagnosis of ASD is available. Further, available medications only manage some symptoms of the disease and have adverse side effects in children. Therefore, there is a need for accurate molecular diagnostic tools to aid in molecular detection and treatment of ASD. To this end, a better understanding of the underlying molecular mechanisms that link ASD etiology to ASD-related behavior is crucial. While genetic factors contribute to syndromic ASD, most cases of ASD are idiopathic with unknown causes, influenced by a combination of epigenetic and environmental factors. TrkB and its downstream signaling pathways, such as Akt and Erk, are hyper-activated in syndromic ASD and hypo-activated in idiopathic cases. Therefore, drugs like rapamycin that inhibit the mTOR pathway downstream of TrkB are beneficial for syndromic ASD but not idiopathic cases. Additionally, insulin-like growth factor 1 (IGF-1), which mitigates ASD-related synaptic disruptions via Akt and Erk signaling, shows unchanged mRNA and protein levels along with its receptor in the idiopathic ASD fusiform gyrus. In ASD with either genetic or epigenetic/environmental causes, disruptions in synaptic connectivity are observed. Synaptic function is regulated by signaling pathways involving brain-derived neurotrophic factor (BDNF) and its receptor, tropomyosin-related kinase B (TrkB), as well as their downstream signaling cascades such as MAPK and Akt. The existing literature suggests that there is an association between BDNF and TrkB signaling pathways and ASD. However, a serious gap in knowledge about the precise molecular role of TrkB in ASD pathology is that our current understanding is correlational in nature and based on observational studies that lack causal experiments. This underscores the importance of further research to understand the causative role of TrkB and its related molecular events in idiopathic ASD. The present work aims to provide a deeper understanding about the causative role of molecular mechanisms underlying TrkB signaling in ASD. ASD mouse models exhibit behaviors and molecular features resembling those observed in human ASD. Therefore, these mouse models are helpful tools for studying ASD. However, understudied physiological confounding factors, such as maternal age and parity, can introduce biases and add to data variability, thus negatively impacting the reproducibility and translational value of ASD mouse models. To achieve a reliable mouse model of ASD, we conducted our first study that examines the impact of maternal age and parity on pregnancy complications, neurodevelopment, and social behavior in mice. Results demonstrate that older maternal age and prior motherhood interact to ensure a normal, steady developmental rate and provide protective effects against anxiety, social impairment, and olfactory deficits. Given the current lack of clarity regarding the causative impact of TrkB on ASD pathology, our subsequent investigation sought to establish a causal relationship between TrkB signaling and ASD. We used the TrkB agonist, LM22A-4 treatment in a validated ASD mouse model. Our results demonstrate that treatment with LM22A-4 effectively rescues the core symptoms associated with ASD (social impairment and repetitive behavior). These findings indicate that impaired TrkB signaling is responsible for ASD-like behavior of valproic acid (VPA)-exposed mice. However, unlike TrkB-related molecular events occurring in the fusiform gyrus of idiopathic ASD, TrkB isoform protein levels, BDNF species, Akt, and Erk total protein levels and activation remained unchanged in VPA-exposed cortices compared to healthy control mice. Since our VPA mouse model does not replicate human idiopathic ASD, our study cannot draw a conclusion on how disruptions in these signaling pathways may contribute to the development and manifestation of ASD symptoms. Cortex is responsible for various aspects of social behavior that are impaired in ASD. However, regulatory mechanisms that are involved in ASD upstream of cortical TrkB and BDNF are not well known. BDNF expression is highly cell-and tissue-specific and is regulated by different sets of transcription factors in specific tissues. While NURR1, the BDNF regulator in midbrain neurons, is associated with ASD pathology, its specific role in regulation of cortical BDNF is not yet well-established. Our third study aimed to understand the role of NURR1 in regulating BDNF specifically in the cortex. We showed that in resting and depolarized neurons, when NURR1 is knocked down, BDNF mRNA levels remained unchanged, suggesting that NURR1 does not regulate BDNF in cortical neurons and highlighting the tissue-specificity of BDNF regulation. In summary, we address the understudied effects of maternal factors on mouse models, which enhances the reliability of ASD research. Further, our studies significantly enhance the understanding of ASD by elucidating the role of TrkB and its downstream signaling pathways in the behavioral aspects of the disorder. We also contribute to the knowledge of BDNF regulation in the cortex, a brain tissue with crucial roles in various aspects of social behavior. In a forward-looking approach, the results of our studies provide valuable insights into mouse modeling of idiopathic ASD and the potential role of TrkB in ASD behavioral symptoms. / Thesis / Candidate in Philosophy / Autism spectrum disorder (ASD) is a condition that is accompanied by challenges in social interaction and repetitive behaviors. ASD is a complicated condition because we do not fully understand all the details of how it works in the body. Studying ASD is important as it is the most challenging condition in children and it is becoming more common, especially in the last two decades. While scientists are developing molecular tools to improve ASD diagnosis and understand its biology, these tools are not widely used in clinics for ASD diagnosis yet. Also, the approved medications available can only help with managing some of the behavioral symptoms like self-harming behavior. Despite the pressing need to find a solution, our recent advancements have not yet brought us closer to a cure for ASD, mainly because of the complexity of the disorder. Therefore, identifying the specific ASD-related mechanisms at the molecular level that contribute to ASD-related behaviors is crucial for gaining a deeper understanding of the disease. In ASD, there are problems with how brain cells communicate with each other. This communication is controlled by certain molecules in the brain, such as brain-derived neurotrophic factor (BDNF) and its receptor, tropomyosin-related kinase B (TrkB), along with other molecules. There is evidence suggesting a link between these molecules and ASD, but we have not fully understood their precise roles because most of the current knowledge is based on observations and correlations, rather than on establishing cause-and-effect relationships. To bridge this gap, our research focused on understanding TrkB's role in ASD. We required reliable mouse models. Since we aimed to induce ASD-like behaviors in mice using an ASD-causing chemical, it was crucial to ensure they were healthy beforehand. We needed to confirm that any social deficits or repetitive behaviors were not due to other factors, such as adverse infancy experiences or impaired interactions between mother and infant. We discovered that sexually mature dams aged between 3 to 6 months, with a history of previous pregnancies and motherhood, give birth to healthier litters. These litters can serve as a more dependable source for our animal behavioral studies. Many cases of ASD in humans are caused by non-genetic factors such as environmental influences like pesticides, air pollution, and the use of certain drugs during pregnancy. In cases of human ASD triggered by non-genetic factors, there is an increase in proBDNF, the precursor of BDNF. However, this proBDNF does not efficiently convert to BDNF. With insufficient BDNF and TrkB receptors, molecules like Akt (protein kinase B, also PKB) and Erk (Extracellular Signal-Regulated Kinase), which are crucial for neuron communication, are also less active downstream. This imbalance disrupts neuron connections, leading to ASD behaviors. In our research, the ASD-causing chemical which we used is valproic acid. It is originally an anti-seizure medication. When pregnant women took valproic acid, the chance of their child having ASD increased. Scientists used this information to inject pregnant mice with valproic acid, and as a result, all the offspring showed ASD-like behaviors. We anticipated that by isolating the brains of these offspring and measuring protein levels of BDNF, TrkB, Akt, and Erk, we would observe a similar pattern to that seen in humans with non-genetic ASD cases. We focused on studying the cortex, a region of the brain responsible for regulating social behaviors in both mice and humans. Since ASD is associated with challenges in social behaviors, we isolated the cortex from mouse brains to analyze protein levels. A chemical known as LM22A-4 with a structure resembling BDNF can bind to TrkB and activate it. We expected that the offspring of pregnant dams injected with valproic acid, which led to reduced TrkB axis activation in their brains, would show improvement in ASD behavior. This anticipation stems from the understanding that LM22A-4 activates the TrkB axis, thus compensating for its reduction, which is thought to be causing ASD-like behaviors. The offspring of mothers injected with valproic acid exhibited ASD-like behaviors, unlike the control mice. Control mice were offspring of pregnant dams injected with a solution containing only the substances used to dissolve valproic acid, typically water and salt (saline). Mice prenatally exposed to valproic acid (VPA) exhibited ASD-like behaviors, but treatment with LM22A-4 helped alleviate these behaviors, promoting more typical behavior patterns. LM22A-4, by activating TrkB receptors, helped to protect the brain from harm caused by exposure to valproic acid before birth. This could mean that valproic acid-induced changes in TrkB-related molecular mechanisms are involved in social behavior difficulties and increased repetitive behaviors seen in autism. Nevertheless, the levels of TrkB, BDNF, proBDNF, Akt, and Erk in the cortex of offspring from mothers injected with valproic acid were like those in the offspring from mothers injected with the saline solution. Therefore, the BDNF and TrkB signaling pathways remained unchanged in the cortex of our valproic acid model in this study, and they differ from those observed in human idiopathic ASD. We also speculated that a protein, called NURR1 acting upstream of BDNF and TrkB might be involved in the process. NURR1 acts as a regulatory protein that binds to the BDNF, increasing the production of copies from the BDNF. We also used a small RNA that targets a specific region in the Nurr1 and inhibits its protein production We anticipated a reduction in Nurr1 levels. As NURR1 acts as an upregulator of BDNF, lower levels of Nurr1 would result in decreased BDNF production. Activating NURR1 resulted in increased BDNF mRNA levels. However, when NURR1 was reduced, BDNF mRNA levels remained unaffected. This led us to conclude that if NURR1 levels decrease, other proteins may step in to maintain BDNF mRNA levels. Therefore, in the cortex, unlike in some other brain regions, the presence of NURR1 is not essential for regulating Bdnf. In summary, before inducing ASD-like behavior in mice using valproic acid, it is crucial to ensure the health of the mice. We used sexually mature mothers with prior pregnancy experience to provide a healthy baseline. We showed valproic acid induced ASD-like behaviors in mice offspring. We also observed that LM22A-4 treatment alleviated ASD-like behaviors of offspring. In our study, we demonstrated that the levels of BDNF, TrkB, Erk, and Akt proteins in the cortex of mice exposed to valproic acid were not affected. For this reason, our mouse model does not resemble human non-genetic ASD. Finally, NURR1's role in BDNF regulation varies by brain region. Lowering NURR1 did not affect BDNF mRNA levels, suggesting compensatory mechanisms. Our findings suggest new directions for further research to better understand the roles of TrkB and BDNF in non-genetic ASD. Overall, this study provides valuable knowledge that can contribute to advancing our understanding of idiopathic ASD-related molecular mechanisms.

Ce mémoire présente mes travaux ayant menés au développement d’une première génération de radioligands marqués au fluor-18 (t1/2 = 110 min) et au carbone-11 (t1/2 = 20.4 min) destinés à l’imagerie cérébrale in vivo du récepteur tyrosine kinase neurotrophique de type 2 (TrkB) en tomographie par émission de positons (TEP). Ces travaux reposent sur l’identification récente de ligands de TrkB non peptidiques à hautes affinités dérivés du 7,8-dihydroxyflavone. La synthèse d’une série de dérivés du 7,8-dihydroxyflavone non-radioactifs de même que des précuseurs à l’incorporation du fluro-18 et du carbone-11 a d’abord été effectuée. Partant des précurseurs adéquats synthétisés, la radiosynthèse de deux radioligands, l’un marqué au fluor-18 et l’autre au carbone-11, a été développée. Ces radiosynthèses reposent respectivement sur une 18F-radiofluorination nucléophile aromatique nouvelle et hautement efficace et sur une 11C-méthylation N-sélective. Les radiotraceurs de TrkB ainsi obtenus ont ensuite été évalués in vitro en autoradiographie et in vivo en tant que traceurs TEP dans des rats. L’évaluation des propriétés physico-chimique de même que de la stabilité in vitro des radiotraceurs sont présentées. Partant d’une série d’analogues cristallisés de ces flavones synthétiques, une étude de relation structure-activité a été menée. La combinaison de cette étude, de pair avec l’évaluation in vivo de la première génération de radiotraceurs de TrkB a aussi permis d’investiguer les pharmacophores nécessaires à l’affinité de ces ligands de même que d’identifier des fragments structurels associés au métabolisme des radiotraceurs. La radiosynthèse d’un troisième radioligand de TrkB et son évaluation TEP in vivo de même que la mise en lumière des modifications structurelles utiles au développement d’une seconde génération de radioligands de TrkB avec des propriétés optimisées pour fin d’imagerie TEP sont aussi détaillés. / This thesis describes my contribution leading to the development of the first-generation positron emission tomography (PET) radioligands labeled with fluorine-18 (t1/2 = 110 min) or carbon-11 (t1/2 = 20.4 min) for the in vivo brain imaging of tropomyosin-related kinase B (TrkB). This research follows from the recent discovery of non-peptidic, high-affinity TrkB ligands derived from 7,8-dihydroxyflavone. The synthesis of non-radioactive 7,8-dihydroxyflavone derivatives and radiolabeling precursors amenable to fluorine-18 and carbon-11 incorporation was performed. Two synthesized compounds have been brought forward as precursors for radiolabeling with either fluorine-18 or carbon-11. Radiosynthesis involved either a novel nucleophilic aromatic subsitution with [18F]fluoride, or N-methylation with [11C]methyl iodide or [11C] methyl triflate. The resulting radiotracers were assessed in vitro by autoradiography and in vivo by PET scans of rats. The physicochemical properties and serum stability of these tracers were also evaluated. X-ray crystal structures of a series of synthetic flavone analogues were used as basis for structure-activity relationship (SAR) analysis. In combination with the above in vivo PET evaluation of these compounds, certain pharmacophores were shown essential for ligand binding affinity. In addition, some structural fragments were associated with in vivo ligand metabolism. The development and radiosynthesis of a third TrkB radiotracer, along with its in vivo PET evaluation and structural analysis, is also described here. In all, better understanding of these tracers have led to the design of potential second-generation TrkB ligands with more optimal properties as PET radiotracers.